The liquid chromatograph (LC) has various separation modes including normal phase mode, reversed phase mode, absorption mode, ion exchange mode, and size exclusion mode, and generally an appropriate separation mode is used according to the type of target sample or other factors. However, for a complicated sample such as an enzyme digesting protein mixture, one-dimensional separation using a single separation mode sometimes may not provide sufficient peak separation. As an advanced analysis technique for use in such a case, a technique called multi-dimensional LC is known which uses a combination of two or more separation modes not affected by each other (refer to Patent Literature 1).
In a typical two-dimensional LC, a flow of mobile phase carrying a liquid sample is introduced into a first-dimension column, components in the liquid sample are separated in the first-dimension column, the sample eluted from the first-dimension column during a predetermined fractionation period T is passed through a trap column, and thereby components of the sample are trapped in the trap column. Next, another mobile phase is passed through the trap column, the components trapped as such are eluted in a narrow time band and introduced into a second-dimension column, and components are further separated in the second-dimension column. Then, an eluate from the second-dimension column is introduced into a detector such as a mass spectrometer or UV/visible spectroscopic detector to chronologically detect the components in the eluate. Normally plural trap columns are prepared, and the trap columns for trapping components are switched from one to another at every fractionation period T, allowing the components in the eluate from the first-dimension column to be trapped without omission and sent to the second-dimension column.
Generally, the two-dimensional LC uses columns having different separation characteristics for the first-dimension column and second-dimension column, and various components in the sample to be analyzed are separated properly by the two column stages differing in the separation characteristics. FIG. 5 is an example of a chromatogram created based on the detection data obtained by a two-dimensional LC. In the chromatogram, narrow peaks appearing in sequence along the direction of time (along the abscissa) are components separated by the second-dimension column.
As can be seen from FIG. 5, in the two-dimensional LC, rough separation is performed in the first-dimension column and finer separation is performed in the second-dimension column. Therefore, if a two-dimensional chromatogram is created with retention time in the first-dimension column being taken as the abscissa and retention time in the second-dimension column being taken as the ordinate and with signal strength being represented by a color scale, gray scale, or other methods, a state of two-dimensional separation by two independent columns can be expressed adequately.
When the time (the fractionation period T) allotted to trap components in one trap column is always constant and the time interval at which a detection signal is obtained by a detector is also constant, the number of detection data items obtained within the fractionation period T is always constant. In this case, as shown in FIG. 6, if a sequence of detection data obtained chronologically during the fractionation period T are arranged along the direction of retention time (along the ordinate) of the second-dimension column of a two-dimensional chromatogram, and the sequence of detection data is arranged so that the sequence is shifted one by one in the direction of retention time (along the abscissa) of the first-dimension column at every interval of the fractionation period T, a good two-dimensional chromatogram can be created. However, depending on the type of detector or depending on detection conditions, the time interval at which the detection signals are obtained may not be constant.
For example, when a mass spectrometer is used as a detector, scan measurements are conducted in the mass spectrometer and the signal strengths of all ions are totaled for each scan measurement as detection data at a given measurement time point. Tandem quadrupole mass spectrometers capable of MS/MS analysis are sometimes used as a detector, but some tandem quadrupole mass spectrometers have an auto MSn function in which a precursor ion is automatically selected from a mass spectrum obtained by a scan measurement and then promptly a subsequent MS/MS analysis is perform (Refer to Patent Literature 2). On such a tandem quadrupole mass spectrometer, when auto MSn analysis is performed in the intervals between repeated scan measurements, the scan measurements are not repeated strictly at constant intervals. The reason is as follows. When any ion to be subjected to subsequent MS/MS analysis is found as a result of a scan measurement, MS/MS analysis is then performed, causing a delay in the start of next scan measurement accordingly. But when no ion to be subjected to MS/MS analysis is found as a result of a scan measurement, the next scan measurement is performed immediately. In another case, when plural ions to be subjected to MS/MS analysis are found, MS/MS analysis is performed using each of the ions as a precursor ion, further delaying the start of the next scan measurement and consequently extending the time interval at which detection data is obtained.
When the number of detection data items obtained within the fractionation period T is not constant due to factors such as described above, problems such as described below arise in creating a two-dimensional chromatogram.
Suppose, for example, the fractionation period T is set to 2 [min] and the repetition frequency of scan measurements is approximately 2.5 [Hz]. The repetition frequency is “approximately” 2.5 [Hz] because the repetition cycle becomes longer, as described above, than that at the basic repetition frequency of 2.5 [Hz] if MS/MS analysis is performed in real time by the automatic MSn function. Therefore, when no MS/MS analysis is performed between scan measurements, the number of scan measurements performed within the fractionation period T, i.e., the number of scans, is 120 [sec]×2.5 [Hz]=300. However, when MS/MS analysis is performed in an interval between scan measurements, the number of scans may be, for example, 298, which is less than 300. Also, as a result of a scan measurement, when a large number of precursor ions are selected and MS/MS analyses are performed corresponding number of times, scan measurement intervals are extended further, resulting in a still smaller number of scans. That is, during data collection by two-dimensional LC, even if the fractionation period T is constant, the number of detection data items obtained within the fractionation period T is indeterminate.
On the other hand, in creating a two-dimensional chromatogram, a predetermined number of detection data items obtained with the passage of time are arranged along the ordinate of a graph. For example, in the above example, the standard number of scans within the fractionation period T is 300. If a shift by the fractionation period T along the abscissa is repeated every 300 detection data items arranged along the ordinate, a two-dimensional chromatogram can be created. An example of a two-dimensional chromatogram created in this way is shown in FIG. 8. The ordinate represents the number of scans, which is equivalent to the number of detection data items. However, as described above, when the number of detection data items obtained within the fractionation period T is, for example, 298 or 297 rather than 300, the data lined up vertically on the two-dimensional chromatogram shown in FIG. 8 does not correctly reflects the state of separation in the second-dimension column. That is, some data items on the upper end side of the vertical line belong to the data in the next fractionation period while some data items on the lower end side of the vertical line are missing.
FIG. 7 is a two-dimensional chromatogram created by arranging 298 detection data items along the ordinate using the same detection data as the two-dimensional chromatogram shown in FIG. 8. Comparing FIG. 7 and FIG. 8, it is apparent that they show quite different states of two-dimensional separation. It can be seen that the peak located in the lower part (the part of shorter retention time in the second-dimension column) in FIG. 7 is relocated in the upper part in FIG. 8. When two such two-dimensional chromatograms are presented, it is difficult for an analyst to determine which of the two reflects a more appropriate state of separation. In any case, with the conventional technique for creating a two-dimensional chromatogram, when the number of data items obtained within the fractionation period T is indeterminate, an inappropriate image which does not correctly reflect the state of separation in an actual column can be obtained.